RESIDUES IN P-GLYCOPROTEIN CATALYTIC SITES THAT REACT WITH THE INHIBITOR 7-CHLORO-4-NITROBENZO-2-OXA-1,3-DIAZOLE

7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-CI) is a specific covalen t inhibitor of P-glycoprotein ATPase activity (M. K. Al-Shawi, and A. E. Senior, 1993, J. Biol. Chem. 268, 4197-4206), Complete inhibition o ccurs at a reaction stoichiometry of 1 mol NBD/mol P-glycoprotein, and the reagent has proved valuable in understanding catalytic mechanisms , particularly in relation to catalytic site cooperativity (A. E. Seni or, and S. Bhagat, 1998, Biochemistry 37, 831-836), The actual locatio n of reaction in the amino acid sequence has not yet been determined. Using a combined mutagenesis and biochemical approach we establish her e that the initial reaction of NBD-CI is with Cys within the Walker A consensus sequence of the N- or C-terminal nucleotide site (Cys-431 or Cys-1074 of human P-glycoprotein). Reaction with either Cys yields fu ll inhibition. It was further found that inhibition consists of dithio threitol (DTT)-reversible and DTT-irreversible components, The former predominates at low pH and the latter at higher pH, This demonstrates that, at higher pH, intramolecular transfer of NBD from Cys to Lys occ urs, probably to the proximate Walker A Lys (Lys-433 or Lys-1076 of hu man P-glycoprotein). After transfer of NBD to Lys, P-glycoprotein ATPa se remains fully inhibited. (C) 1998 Academic Press.